Interaction of MHV RNA with mtHSP70 and m-aconitase

MHV RNA 与 mtHSP70 和 m-乌头酸酶的相互作用

• 批准号: 6760101
• 负责人: JULIAN L LEIBOWITZ
• 金额: $ 32.74万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6760101
• 关键词: Interaction; MHV; RNA; mtHSP70; aconitase

DESCRIPTION (provided by applicant): We have recently identified four proteins which interact with a protein binding element [3'(+)42 element] contained within the last 42 nucleotides of the mouse hepatitis virus genome. In this application we propose to examine the interaction of these four proteins, mitochondrial HSP70 (mtHSP70), mitochondrial aconitase (m-aconitase), HSP60, and HSP40, with MHV RNA. We will employ fluorescence resonance energy transfer (FRET), immuno-electron microscopy, and cell permeant cross-linking studies to verify that the interaction of these proteins with MHV RNA takes place in intact cells. We will undertake a series of biochemical studies to determine if the MHV RNA interacts with mtHSP70, m-aconitase, and HSP60 prior to the importation of these proteins into mitochondria, or alternatively, if these MHV RNA interacting proteins are exported from mitochondria. We will also determine if mitochondrial function is altered during MHV infection. To better characterize the structural basis for these interactions we will perform a series of experiments to map the residues of m-aconitase, mtHSP70, HSP60, and HSP40 which are in contact with the MHV 3'(+)42 protein binding element. We will subsequently carry out a series of mutagenesis experiments to map residues of these proteins required for RNA binding. A second line of mutagenesis experiments will more precisely define the sequence requirements of the RNA for protein binding. We have determined that these proteins interact during binding, thus dominant negative mutants will be particularly sought. We have determined that mtHSP70 is tyrosine phosphorylated, and that the phosphorylation state of the MHV 3'(+)42 binding proteins regulates their binding the MHV 3'(+)42 element. We will perform a series of pharmacologic and genetic manipulations to investigate the role of tyrosine phosphorylation in regulating the interaction of these proteins with MHV RNA. The functional significance of the interaction of mtHSP70, m-aconitase, HSP60, and HSP40 with MHV RNA on virus replication will be examined in a series of experiments employing mutational and over-expression strategies.

描述（由申请人提供）：我们最近鉴定了四种与小鼠肝炎病毒基因组最后42个核苷酸内包含的蛋白质结合元件[3'(+)42元件]相互作用的蛋白质。在此应用中，我们建议检查线粒体 HSP70 (mtHSP70)、线粒体乌头酸酶 (m-乌头酸酶)、HSP60 和 HSP40 这四种蛋白与 MHV RNA 的相互作用。我们将采用荧光共振能量转移 (FRET)、免疫电子显微镜和细胞渗透交联研究来验证这些蛋白质与 MHV RNA 的相互作用发生在完整细胞中。我们将进行一系列生化研究，以确定 MHV RNA 在将这些蛋白质输入线粒体之前是否与 mtHSP70、m-乌头酸酶和 HSP60 相互作用，或者这些 MHV RNA 相互作用蛋白质是否从线粒体输出。我们还将确定 MHV 感染期间线粒体功能是否发生改变。为了更好地表征这些相互作用的结构基础，我们将进行一系列实验来绘制与 MHV 3'(+)42 蛋白结合元件接触的 m-乌头酸酶、mtHSP70、HSP60 和 HSP40 残基的图谱。我们随后将进行一系列诱变实验来绘制 RNA 结合所需的这些蛋白质的残基图。第二线诱变实验将更精确地定义蛋白质结合的 RNA 序列要求。我们已经确定这些蛋白质在结合过程中相互作用，因此将特别寻找显性失活突变体。我们已经确定mtHSP70是酪氨酸磷酸化的，并且MHV 3'(+)42结合蛋白的磷酸化状态调节它们与MHV 3'(+)42元件的结合。我们将进行一系列药理学和遗传操作来研究酪氨酸磷酸化在调节这些蛋白质与 MHV RNA 相互作用中的作用。 mtHSP70、m-乌头酸酶、HSP60 和 HSP40 与 MHV RNA 相互作用对病毒复制的功能意义将通过一系列采用突变和过表达策略的实验进行检验。